Prevention of Enzymatic Browning in Fruits and Vegetables

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Chapter 4

Prevention of Enzymatic Browning in Fruits and Vegetables A Review of Principles and Practice 1

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Lilly Vámos-Vigyázó

Central Food Research Institute, P.O. Box 393, H-1536 Budapest, Hungary

Enzymatic browning of fruits and vegetables has not ceased to be a problem for processors, although the underlying basic reactions have been known for a long time. One of the difficulties in selecting the mode of browning prevention consists in the necessity of complying with food safety regulations, while at the same time taking into account the marketability of the product as affected by taste andflavor,texture, etc. The ever increasing amount of literature accumulated on the topic shows great progress in establishing the phenolic composition of foods, enzyme purification and the introduction of new browning inhibitors. This review gives an account on the latest achievements in the prevention of browning along with some of the author's earlier results.

In the early 1980's this author had her first opportunity to review the literature on the polyphenol oxidase (PPO)-catarysed transformation of endogenous phenols in raw food material to - in most cases undesirable - brown pigments (7). Since that time the interest in the topic has not slackened. On the contrary, the field of research has become even broader. Beside enzymic browning offruitsand vegetables, which is still of interest, an increasing number of papers have been devoted to enzymatic discoloration of cereals, oilseeds and sugar cane (2-6) as well as to commodities of animal origin (seafood) (7,5). The latter are of particular interest as, e.g. lobster PPO is present in a latent form in the tissues (7-9), a phenomenon rarely encountered in the plant kingdom. This review tries to give an overview on the prevention of enzymatic browning infruitsand vegetables mainly based on the literature of the 1990's. Current address: Szentkirályi utca 29-31, H-1088 Budapest, Hungary

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0097-6156/95/0600-0049$12.00/0 © 1995 American Chemical Society In Enzymatic Browning and Its Prevention; Lee, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Principles of the Prevention of Enzymatic Browning The principles of browning prevention have not changed with time and are essentially the same as those applying to the inhibition of any tissue enzyme, i.e.:

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a/ inhibition/inactivation of the enzyme b/ elimination/transformaton of the substrate(s) c/ combination of a/andb/. In the light of recentfindingsit is, however, not easy to classify an inhibitor or an inhibitory process as belonging exclusively to one of the three categories. Many inhibitors act both on the enzyme and on its substrates. For all the three cases mainly substances or procedures compatible with food safety and marketability requirements shall be dealt with here. Prevention does not necessarily consist of post-harvest treatment only. Much can be done to reduce browning occurring during storage or processing by selecting cumvars of slight browning tendency (70) and by appropriate agricultural techniques (77). Studies into the changes in browning tendency, PPO activity and phenolic composition and concentration during ripening (12, 13) might be equally helpful to the grower and the processor, although harvest times can hardly be adjusted to the optimum values of these parameters only. A vast amount of data has been accumulated during the past decade on the characteristics of PPO in different commodities (14-23). As methods of enzyme extraction and purification improved simultaneously, findings often are not in agreement with those published earlier for the same commodity. The same applies for the research into the phenolic composition of foods (24-26). Here the outstanding importance of HPLC should be stressed. From the aspect of enzymatic browning and its inhibition research into the fate of phenolic substrates during PPO-catarysed oxidation might be of outstanding importance (27,28). Enzyme inhibition can be reversible or irreversible. The latter is often achieved by physical (heat) treatment, while chemicals might act in one or the other way. Prevention of Enzymatic Browning with Chemicals Recent Uses of Traditional Browning Inhibitors. Most recent work on the prevention of enzymatic browning is aimed at replacing sulfite (29, 30). Sulfur dioxide, sulfites, bisulfates and metabisulfates inhibit enzymic and nonenzymic browning (37) and are effective against microbial infection. However, owing to their harmful effect on health their use has been restricted or banned altogether in several countries. It seems difficult to substitute them in vinification (32-34). [Some success has been achieved, though, with ascorbic acid or ascorbic acid containing mixtures of chemicals (34)]. As shown with metabisulfite, this class of inhibitors act a/ on the quinones formed by PPOcatalysed oxidation of o-dihydroxy phenols and b/ on the enzyme itself by irreversibly binding to the "met" and "oxy" forms of binuclear copper at its active site (35).

In Enzymatic Browning and Its Prevention; Lee, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Some recent uses of classical browning inhibitors other than sulfite are summarized in Table I. Table L Recent Uses of "Classical" Browning Inhibitors in Fruits and Vegetables Reference Remark Commodity Inhibitors) Concentration 36 Apple Heating (60-70C, 15 Ascorbic acid 10 (g/1) (slices) min) or decreasing of pH promoted inhibition 37 Apple Ascorbic acid + 10 AA+2 CA Dip (5 min) (cubes) citric acid (g/i) (Golden Delicious) 37 Apple Dip (5 min) Ascorbic acid + 10 AA (cubes) NaCl + 0.5 NaCl (Golden (g/i) Delicious) 38 Apple; Infiltration; ΑΑΡ was Ascorbic acid potato hydrolysed by or ascorbic acid endogenous acid phosphate phosphatase 40 Potato Effective in extending Ascorbic acid + 40 + or 25 + storage life of abrasion-, citric acid + 10+ 10 + sodium acid 10+ 10 + rye- or high pressure steam-peeled potatoes pyrophosphate after digestion and 2 2+ + CaCl +AAP (Mg-sah) + 16 + removal of digested surface prior to applying A A P (Na-sah) 5-min dip 15 (β/1) 41 Garlic Effective during storage Citric acid 10 (g/i) (chopped) at4°C 42 Avocado 100% inhibition L-cysteine 0.32 mM 42 Banana 100% inhibition L-cysteine 5.0 mM AA: ascorbic acid; ΑΑΡ: ascorbic acid phosphate; AAP : ascorbic acid triphosphate; CA: citric acid. 2

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From the data tabulated it can be seen that ascorbic acid is a browning inhibitor still much dealt with. Its action can be enhanced by citric acid and NaCl in concentrations that are ineffective in themselves or even increase enzyme activity. (It is interesting that an increase in activity was noted also for ascorbic acid alone up to the concentration given in the Table) (38).

In Enzymatic Browning and Its Prevention; Lee, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Ascorbic acid phosphate (ΑΑΡ) might act as an ascorbic acid "reservoir", when the browning tendency of the product is not too strong. Otherwise the AA released by endogenous ascorbic acid phosphatase might be soon depleted. A reduction of the inhibitor solution pH to 2 inhibits ascorbic acid phosphatase and makes the effect of ΑΑΡ safer. Reduced pH and the use of ascorbic acid-2-triphosphate provide for a gradual release of ascorbic acid (39). The question arises here to what extent lowering of the pH contributes to the inhibition of browning and if this pH is compatible with the sensory and technological properties of the given commodity. Another group of "classical" browning inhibitors are sulfur-containing amino acids and peptides or alcohols with L-cysteine as prototype. The action of cysteine is complex. It forms addition compounds with phenolic substrates. The structure of some of these compounds is now well established, e.g. 5-S-cysteinyl-3,4-dihydroxytoluene is formed from 4-methylcatechol and 2-S-cysteinylchlorogenic acid from chlorogenic acid (43). Cysteine also forms adducts with quinones. The fate of the quinones and the adducts (and the efficiency of browning inhibition by cysteine) was found to depend on the ratio thioliphenol and also on the pH. Cysteine-quinone adducts proved to be competitive inhibitors of PPO. They enter enzymatic as well as non-enzymatic oxidation reactions with the quinones, whereby phenols are regenerated. These may undergo enzymatic oxidation causing color formation. In order to prevent discoloration, the relative cysteine concentration must be sufficient to transform all the substrate into colorless adducts (44, 45). N-acetyl-L-cysteine and reduced glutathione were found to be even more potent browning inhibitors than L-cysteine (for apples and potatoes) (46), while dithiothreitol, a reversible inhibitor of PPO was much more efficient than glutathione (for mushrooms) (47). The efficiency of cysteine (and also of aromatic amino acids) and the type of inhibition were found to depend, apartfromthe nature of the inhibitor, also on the method used for determining activity (polarographic or spectrophotometry) (48, 49). Some Earlier Results with Traditional Inhibitors from the Author's Laboratory. Some of the author's own work with inhibitors used to prevent browning of apples and peaches is shown in Tables Π and m. (Vàmos-Vigyàzo and Gajzago, Central Food Research Institute, Budapest, unpublished data). The discoloration was measured by a reflectance method (50, 51) using slices of Starking and Golden Delicious apples as well as Elberta and Ford peaches treated and not treated with inhibitors, respectively. The wavelengths used for reflectance measurements were 540 nm for apples, 470 nm for the white-fleshed peach cumvar Elberta and 580 nm for the yellow-fleshed peach cultivar Ford. Fruit slices were immersed at room temperature (22-24 °C) into inhibitor solutions of various compositions and concentrations for various times. The progress of discoloration was read from the scale of the spectrocolorimeter "Spekol" (Zeiss, Iena, Germany). Readings were performed at least up to 10 min, during the first 2 min every 30 s and later every 60 s. From the linear parts of the saturation curves obtained the "initial browning rate" (BA) of the samples was calculated by linear regression. The number of replicates was 4-7. Mean BA values and standard deviations were calculated. The BA-

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values of inhibitor-treated samples were subtracted from the respective values of the untreated controls, related to the control values and expressed as % inhibition. If no discoloration occurred up to 30 min, inhibition was considered to be 100%. The statistical evaluation of the results was performed by Student's /-test (comparing the BA-s of the inhibited with the respective control sample). The results obtained with apples are summarized in Table Π. Table Π. Inhibition of the Discoloration of Apple Slices by Dipping into Various Inhibitor Solutions Inhibition Apple cultivar Dipping time Inhibitor and concentration (%) (min) (%) 0* 1 Starking 3 CA(2) 57 ±10 5 57 ±23 1 60 ±20 Starking CA(4) 3 80 ±18 5 100 1 73 ±17 Starking CA(6) 3 90 ±13** 5 100 1 90 ± 7 Starking AA(1) 3 98 ± 3** 5 100 1 95 ±13** Starking AA(2) 3 99± 4** 5 100 100 Starking 3 A A ( l ) + CaCl (0.1),pH8.1 0 1 Starking 3 CnA (0.0075) 78 ± 8 5 80 ± 8 0 1 Starking 3 CnA (0.015) 80 ±10 5 83 ± 8 1 5 ±30 Golden Delicious 3 CnA (0.0075) 84 ± 8 100 5 1 5 ±30 100 Golden Delicious 3 CnA (0.015) 100 5 CA: citric acid; AA: ascorbic acid; CnA: cinnamic acid; * statistically non-significant increase; ** not significantly differentfrom100%. 2

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With Starking apple slices the shortest dipping times and the lowest inhibitor concentrations yielding complete inhibition were 5 min in 4% (0.2 M) citric acid, and 5 min in 1% ascorbic acid (0.057 M), respectively. (Some of the other values were not significantly different from 100 inhibition either). Dipping time could be reduced to 3 min by adding 0.1% (9 mM) CaCl to the above ascorbic acid solution. CaCl is known to have a beneficial effect on apple flesh firmness. Cinnamic acid could be applied only at very low concentrations (0.5 and 1 mM) as it imparted an off-taste to the fruit, described by the panelists as "metallic". Complete inhibition of the browning of Starking apple slices could not be achieved with either concentration. A prolongation of the immersion time from 3 to 5 min had no significant effect on the efficiency of this inhibitor. With Golden Delicious, the cultivar less susceptible to browning, complete inhibition could be achieved by applying 0.5 mM for 5 or 1 mM cinnamic acid for 3 min. The results obtained with peach slices are shown in Table ΙΠ.

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Table ΠΙ. Inhibition of the Browning of Peach Slices by Dipping into Various Inhibitor Solutions Peach cultivar Relative Inhibitor and Dipping time concentration (%) min Inhibition % Ε ΑΑ(1) 1 85 ±33* Ε 100 1 ΑΑ(3) F 100 1 Ε 1 CA(1) 30 ± 6 Ε 1 CA(3) 39 ± 12 Ε 2 42 ± 13 Ε CA(5) 2 71 ± 19 Ε Sucrose (30) 2 64 ± 8 F 1 82 ± 21* Ε Sucrose (50) 2 62 ±10 Ε 100 Sucrose (30) + CA (3) 2 Ε 100 Sucrose (50) + CA (3) 2 Ε NaHS03 (0.1) 100 2 E: Elberta; F: Ford: AA: ascorbic acid; CA: citric acid *: not significantly different from 100% inhibition. Complete inhibition of browning could be achieved with a 1-min dip in 3% ascorbic acid for both cultivars tested. Citric acid was only tried with the cultivar Elberta and did not yield complete inactivation with any of the concentrations and dipping times tried. No complete inhibition could be achieved either with sucrose solutions alone. However, both combinations of sucrose with citric acid gave full protection against browning. The joint action of these two inhibitors seems to be additive. Finally,

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complete inhibition of browning of Elberta peaches could be achieved with a 0.1% solution of sodium bisulfite.

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Recently Developed Inhibitors of Enzymatic Browning. The past years have witnessed a boom in the development of new browning inhibitors. Part of them are now available on the market. Some of them are listed in Table IV along with their field of application tried so far. Aromatic Compounds. Aromatic carboxylic acids and substituted phenols (29, 52) have long been in use as inhibitors of PPO, mainly for kinetic studies of inhibition, but partly also for preventing browning with more or less success. 4Hexylresorcinol (4HR) is one of the recently discovered, patented (53) and approved browning inhibitors of this class. It was first reported to inhibit shrimp black-spotting caused by PPO (54). 4HR was much more effective on the purified mushroom enzyme than on a crude extract and did not inhibit laccase (55). Apple slices (in syrup) required only 1/5 the concentration of 4HR of that necessary for achieving the same result with sulfite (56). Tropolone (2-hydroxy-2,4,6-cycloheptatrien-l-one) is a copper chelator slowly binding to the "oxy" form of the enzyme (58). It was found to be a substrate of horseradish peroxidase in the presence of H 0 and is, therefore, assumed to be helpful in distinguishing this enzymefromPPO (59, 60). Kojic acid [5-hydroxy-2-(hydroxymethyl)-y-pyrone] interacts with o-quinone formation from o-diphenols by decreasing 0 uptake by the enzyme. It inhibits also the monophenolase activity of PPO (62). 2

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Glucosidated Substrates. (+)-Catechin 3-O-a-D-glucopyranoside obtained from (+)-catechin by different enzyme-catalysed reactions (with cyclodextrin glucanotransferase, E.C.2.4.1.19 and soluble starch, and with a bacterial sucrose phosphorylase, E.C.2.4.1.7, respectively), proved to be a strong inhibitor of mushroom PPO (63, 64). As other PPO-substrates can be transformed by glucosidation to inhibitors as well (64), this group of compounds might be of interest for future research. Proteolytic Enzymes. The possibility of an enzyme, itself a protein, being attacked and inactivated by proteolytic enzymes is more than obvious. In spite of this, protease preparations have not been systematically tried for a long time as inhibitors of PPO and enzymatic browning. The observation that the contact with kiwi slices or puree inhibits enzymatic browning of otherwise susceptible commodities, drew the attention to this group of compounds. [Kiwi fruit is known to contain a highly active protease (actinidine, 65, 66)]. Of the proteolytic enzymes tested so far mainly three plant proteases (ficin from figs, papain from papaya and bromelain from pineapple) proved to be effective. All the three proteases are sulfhydryl enzymes of broad specificity (66).

In Enzymatic Browning and Its Prevention; Lee, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

In Enzymatic Browning and Its Prevention; Lee, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

Carbon monoxide Hypochlorites

Maltodextrins Honey

Carrageenans

(+)-Catechin 3O-cc-D-ghicopyranoside Proteases

Kojic acid

Tropolone

Name of inhibitor 4-Hexylresorcinol

Na- and Ca-saks

w

ficin, papain, bromelain (sulfhydryl enzymes) sulfated polysaccharides oligosaccharides Peptide of M 600

the relative efficiency of the enzymes depends on treatment temperature and on the commodity

apple, potato slices

mushroom PPO

71 72 dips containing low concentrations (17.5-140 ppm) of inhibitor

69 70

67, 68

66

63, 64

61, 62

57, 58

55, 56

Reference

reversible inhibition of catecholase activity

+

synergistic effect with citric acid; long-lasting inhibition apple juice and dice K -ions enhance inhibition ground apples apples, grape juice

apple and potato slices

Table IV. Some Recently Developed and/or Tried Browning Inhibitors Remark Chemical nature Field of application substituted phenol mushroom, 90% inhibition achieved with 100 μΜ (crude apples, etc. mushroom PPO); delay of onset of browning obtained with 200 μΜ at 25 °C (apple slices in syrup) mushroom and cycloheptatriene causes biphasic inhibition at up to 30 μΜ concentration; derivative grape PPO both initial and constant rate of reaction depend on inhibitor concentration especially efficient at inhibiting enzymatic L-DOPA mushroom, apple, γ-pyrone potato, shrimp, oxidation derivative spiny lobster mushroom

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Carbohydrates. A number of carbohydrate derivatives were found to be effective in preventing enzymatic browning of foods. Carrageenans, a group of naturally occurring sulfated polysaccharides, but also amylose sulfate and xylan sulfate were reported to inhibit, in low concentrations (less than 0.5%), browning of unpasteurized apple juice and diced apples. The inhibitory effect could be synergistically enhanced by citric acid (0.5%) (67, 68). The inhibitory effect of makodextrin (DE 10) was enhanced by K and assumed, therefore, to be related to pyruvate kinase in apples (69)

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+

Peptides. PPO activity and browning were found to be inhibited by honey. Experiments with model solutions showed PPO inhibition to be non-competitive with (-)-epicatechin as substrate and enzyme activity could be progressively inhibited with time by preincubation in solutions of honey (70). The inhibitory effect was due to a peptide of MW 600. Carbon Monoxide. CO gas atmosphere was found to inhibit mushroom PPO reversibry, whereby it prevented self-inactivation of the enzyme (71). The latter feature of this inhibitor might be useful in studies on PPO action. However, CO - as a substance harmful to human health - does not seem to be of any practical use in preventing browning of food materials. Hypochlorite. Sodium and calcium hypochlorites in low concentrations (down to 17.5 ppm) were reported to inhibit enzymic browning of green beans, apples and potatoes. These compounds obviously act on the enzyme protein (72). It is, however, improbable that this potent disinfectant should ever be approved for use in foods. Miscellaneous Browning Inhibitors. A number of browning inhibitors were found by researchers by observation or trial-and-error methods. The chemical nature of these inhibitors is mostly unknown. Good results were achieved with pineapple juice in treating applerings.The juice was treated in different ways, whereby the best results were achieved with a cation exchanged fraction of the original juice. This fraction contained ascorbic acid (0.1 mg/ml), phenolics (0.41 mg/ml) and amino acids (formol value: 0.64 meq/100 ml) and ensured 100% inhibition for at least 12 h. Pineapple juice was effective withfreshapple rings kept in air or vacuum-packed as well as with dried apples. The inhibitor in pineapple juice was found to be a neutral, low-molecular substance (73). A patent has been recently granted for an inhibitor obtained by repeated filtration of a fig latex suspension. The inhibitor is claimed not to contain anyfigprotease (ficin) and to inhibit enzymic and non-enzymic browning, e.g. of mushrooms, wine and shrimps. An MW < 5000 was established for its active principle (74). Acetone solublefractions(ASF) prepared from various fruits (plums, peaches, apples, pears and grapes) were found to act on the PPO activity of these fruits as measured with chlorogenic acid in a selective way. For example, ASFfromgrapes inhibited the enzymefrompeaches, apples and grapes: ASF from plums inhibited PPO

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from peaches and apples, but accelerated the reaction catalysed by the enzyme from pears, plums and grapes, while ASF from peaches and apples showed only a stimulating action (75). Maillard reaction products obtained by heating a solution containing glucose and glycine were found to inhibit PPO and also peroxidase activity (76). Stabilization of Fruit Juices Against Enzymatic Browning. The inhibition of the enzymatic oxidation of endogenous phenols in fruit juices represents a special problem. Non-enzymic oxidation of the phenols in the juice using an air-flow (bubbling) with subsequent removal of the colored macromolecular products by filtration has long been practised by juice manufacturers for obtaining clear and stable products. Recent research has led to the development of a fungal laccase preparation (by fermentation using the white-rot fungus Trametes/Polyporus versicolor, 77). The enzyme immobilized on agarose-based activated matrices was reported to lend itself to the removal of phenolicsfromwhite grape musts and wines. The immobilized enzymereactor could be reused at least 8 times, and showed practically no losses of activity when properly stored (78). Another method of browning control is based on the addition of chitosan to apple or pear juice prior to filtration through diatomaceous earth. For the treatment of Mcintosh apples about 200 ppm chitosan proved efficient, whereas Bartlett and Bosc pears required 1000 ppm. Chitosan proved to be inefficient with very ripe pears and could not be used if the juices were to be centrifuged (79). A recent patent reports on trapping phenols of raw fruit and vegetable juices using soluble or insoluble cyclodextrins. The latter could be used also as column fillings (80). Traditional and Recently Developed Physical Methods for Inhibitng Enzymatic Browning Reports on the use of traditional methods of browning prevention by heat treatment can still be found in the literature. Blanching. Water blanching was used to prevent enzymatic darkening infrozensweet potatoes. Treatment at 100 °C for 3 min or at 94 °C for 5 min gave satisfactory protection against darkening without reducing the phenol levels (8J). Blanching in boiling water was used also for preserving the green color in dried pepper. Longer boiling times (15 min) resulted in better color retention for berries. Microwave heating alone was insufficient and did not yield berries of lighter color as compared to sun-drying. Microwave heating combined with blanching was found to be most efficient for enzyme inactivation and permitted reduced treatment times (82). Ultrafiltration. Enzymic browning of fluids can be reduced by ultrafiltration (UF). A study of wine treatment by UF at different molecular weight cut-offs between 100,000

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and 30,000, before or after fermentation showed the results to be culrrvar-dependent. Browning during storage was found to be dependent also on the headspace in the bottles (83). Sonication. Recent studies have been aimed at combining heat treatment with ultrasonic waves in order to reduce the heat resistance of mushroom PPO. The simultaneous application of heat and ultrasonic waves had a synergistic effect on enzyme inactivation. The efficiency of the procedure was reported to increase with increasing amplitudes of the ultrasonic waves. This manifested itself in the decrease of D values (decimal reduction times at constant temperature). The process named manothermosonication needs, however, special and probably expensive equipment and might be of use, in the first place, with commodities that are damaged by drastic heat treatment (84). Supercritical carbon dioxide. Supercritical carbon dioxide (SC-C0 ) treatment was also tried for inactivating PPO from potato peel,freshFlorida spiny lobster and fresh brown shrimp. The purified enzyme preparations dissolved in a pH 5.3 buffer showed but slight losses of activity (5% for potato peel PPO) when heated at 43 °C for 30 min. Treatment of the enzymes with high-pressure (58 atm) CO2 caused a dramatic loss of activity: after 1 min, the residual activity of potato PPO, the most resistant of the three preparations, was only 45%. However, 30 min were required for an activity loss of 91%. The pH of the potato PPO solution was reduced from 6.1 to 4.1. The treatment caused changes in the isoenzyme composition of all the three PPO-s and compositional changes in secondary structure. These were slightest for the most resistant potato enzyme. The SC-C0 -treated potato-enzyme regained 28% of the original activity in thefirst2 weeks of 6 weeks offrozenstorage. This activity then gradually decreased as time progressed. The pH of the enzyme solution returned to its original value (85). With the exception of ultrafiltration, (which is applicable only to fluids) all the procedures dealt with in this last section also involve heat treatments. This restricts their application to products consumed in the cooked, stewed, fried, etc. state. Moreover, most of them require sophisticated equipment. It seems, especially with fruits, that inhibition with chemicals will play the primary role in the prevention of enzymatic browning also, at least, in the near future. However, as alien additives are considered ever more undesirable in food for health reasons (justified or exaggerated) the search for other methods will, most probably, go on. As the "appropriate agrotechnics" mentioned in the introduction also involve the use of chemicals, these might be banned sooner or later as well. Cultivar selection might remain promising in the future, too, and great hopes might be set on the genetic modification of PPO in various commodities. Research in this direction is in progress in several countries with promising results (see the respective papers in this volume). 2

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Literature Cited 1. Vámos-Vigyázó, L. CRC Crit. Rev. Food. Nutr. 1981,15,49-127. 2. McCallum, J. Α.; Walker, J. R. L. J. Cereal Sci. 1990, 12, 83-96.

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3. 4. 5. 6. 7. 8.

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